Atherosclerosis, the hardening and narrowing of arteries leads to coronary artery disease, a leading cause of death. The progression of atherosclerosis is accelerated by low levels of high-density lipoprotein cholesterol (HDL-C), which is responsible for transporting cholesterol from the bloodstream back to the liver. HDL-C levels are influenced by numerous environmental and genetic factors. Large human genome- wide association studies (GWAS) provide new opportunities to identify novel DNA variants and genes that influence HDL-C and risk of disease. A recent GWAS identified four variants (r2 > 0.75, rs737337) located less than 5 kb upstream of ANGPTL8 that are strongly associated (n~185,000, P=4.6x10-17) with HDL-C. Bioinformatic predictions and experimental data including epigenetic marks, transcription factor binding, and open chromatin sites that correlate with ANGPTL8 expression suggest a regulatory role for these variants acting on ANGPTL8. Studying these variants will help reveal the mechanisms by which ANGPTL8 influences HDL-C levels. Circulating ANGPTL8, also named Betatrophin, is correlated with atherogenic lipid profiles in insulin resistant individuals, and is increased following a meal, suggesting a role in lipid metabolism. ANGPTL8 expression is limited to the liver and adipose tissues despite being entirely contained within one intron of the ubiquitously expressed DOCK6 gene. Given that the liver plays a critical role in the regulation of HDL-C, the expression pattern provides further support that the HDL-associated variants act on ANGPTL8 rather than DOCK6 or other nearby genes. Our goal is to identify the functional molecular mechanism(s) underlying association of this GWAS locus with HDL-C and the allele-specific effects of metabolic perturbations on ANGPTL8 expression. We hypothesize that the tissue-specific expression of ANGPTL8 is mediated by promoter, enhancer, and/or repressor interactions, and that these interactions can be influenced both by HDL-associated genetic variants and by specific metabolic stimuli. We will determine the tissue specificity of the ANGPTL8 promoter and regulatory elements using reporter assays, and we will investigate allelic differences in transcriptional activity and protei binding of HDL-C- associated variants to determine the molecular mechanism at this locus. By engineering cell lines containing the alternate HDL-C-associated alleles, we will evaluate the effects of the variants in a genomic context. To determine the metabolic stimuli that alter cell autonomous expression of ANGPTL8, we will treat the cell lines containing each haplotype with glucose, insulin, and other stimuli suggested to regulate ANGPTL8. Taken together, these experiments will elucidate the molecular mechanisms by which ANGPTL8 is activated, how HDL-C-associated DNA variant(s) in distinct regulatory elements interact, and how metabolic perturbations influence these processes. Studying the human variants and their interaction with metabolic factors will pave the way for developing novel therapies based on this new target gene.